Clinical characteristics of idiopathic interstitial pneumonias with anti-Ro52/tripartite motif-containing 21 antibodies

Antibodies to Ro52/tripartite motif-containing 21 (TRIM21), referred to as anti-Ro52, are found in patients diagnosed with diverse systemic autoimmune rheumatic disease and associated with interstitial lung diseases. However, little is known about the clinical characteristics of anti-Ro52 in patients with idiopathic interstitial pneumonias (IIPs). We aimed to analyze the prevalence, co-existent autoantibodies, and clinical characteristics of anti-Ro52 in patients with IIP. The study enrolled 288 patients diagnosed with IIP. Clinical, laboratory and radiographic findings of IIP patients were compared between anti-Ro52 positives and negatives. Anti-Ro52 (20/288; 6.9%), anti-ARS (18/288; 6.3%), and anti-Ro60/SS-A (16/288; 5.6%) were the most common autoantibodies detected in IIP patients. Among 20 IIP patients who had anti-Ro52, anti-ARS was present in 8 (40%) patients. The criteria for interstitial pneumonia with autoimmune features (IPAF) were significantly better fulfilled by patients with anti-Ro52 than those without (P = 0.001). Meeting serological domain (P < 0.001) and Raynaud’s phenomenon (P = 0.009) were significantly more common in the anti-Ro52-positive patients. Anti-Ro52-positive IIP patients have clinical features consistent with IPAF. Anti-Ro52 may have an important role in detecting the autoimmune phenotype in IIP patients.


Discussion
This is the first study investigating the frequency of serum anti-Ro52 antibodies in unselected patients with IIP. Similar to the prevalence of anti-ARS (6.3%), anti-Ro52 was detected in 6.9% of patients with IIP. Presence of serum anti-Ro52 was significantly associated with fulfillment of IPAF criteria, particularly with respect to the serological domain and Raynaud's phenomenon, in IIP patients. Anti-Ro52 is mostly present in patients with different types of SARD 22 , as seen in nearly half of the patients with Sjögren's syndrome 12 , SSc 15,16 , and SLE 17 and 20-30% of patients with PM/DM 13,14 , In this study, the prevalence of anti-Ro52 (6.9%) in IIP was lower than in SARD but higher than in healthy individuals (< 0.2-1%) 23 . In addition, as in patients with PM/DM 14,18,19 , anti-Ro52 frequently co-existed with anti-ARS. Among the anti-ARS that co-existed with anti-Ro52, anti-Jo-1 found in three cases was the most common, in addition to anti-PL-7, anti-PL-12, anti-EJ, and anti-KS.   www.nature.com/scientificreports/ IPAF criteria (P = 0.001) related to the serological domain (P < 0.001) were more frequently fulfilled by anti-Ro52-positive (50%) than anti-Ro52-negative patients (17%) in our IIP cohort. A previous retrospective study showed that 49.3% of the ILD patients who had anti-Ro52 met the IPAF criteria, similar to our result 21 . Anti-Ro52-positive patients could be negative in immunofluorescence ANA tests, however, anti-Ro52 was associated with IPAF serological domain, indicating that it frequently coexists with the other autoantibodies included in the IPAF serological domain (Table 2). Co-existence of anti-Ro52 with other specific autoantibodies in various SARD have been reported 14,15,18 . Although anti-Ro52 is not specific for a particular type of SARD diagnosis, a 14-fold increased risk of developing SARD was reported in IIP patients who met the IPAF criteria 24 . Thus, presence of anti-Ro52 might be considered as a useful clinical diagnostic tool for the early detection of SARDs in patients with IIP who pose a higher risk of developing in the future.
IPAF criteria are used for the identification of a subset of IIP patients exhibiting autoimmune features but lacking a definitive diagnosis of SARD 5 The ATS/ERS task force has suggested the need for further validation and revision of IPAF criteria 5 . Accordingly, there has been a proposal for the inclusion of several myositis-specific antibodies (anti-NXP-2, anti-TIF1γ) in the IPAF criteria 25 . In contrast, anti-double stranded DNA, anti-Sm, anti-topoisomerase I (Scl-70), and anti-MDA5 are disease-specific diagnostic antibodies that have a proven link to the diagnosis of SLE 26 , SSc 27 , and clinically amyopathic DM (CADM) 28 . These disease-specific marker antibodies are produced prior to the clinical manifestation of the associated SARD and the association of IPAF with these antibodies might be an indication of early stage SARD. The appropriateness of the inclusion of these antibodies in the IPAF criteria is controversial.
Some commercial assays separately measure antibodies to Ro60 and Ro52, while other anti-Ro/SS-A immunoassays use a mixture of Ro60 and Ro52 as antigen. However, recent literature indicates that Ro60 and Ro52/ TRM21 are unrelated molecule and Ro52/TRIM21 is not a part of Ro60/SS-A-hYRNAs complex [29][30][31] . Thus, separate measurement of anti-Ro52 and anti-Ro60/SS-A is recommended because of their biochemical and immunological differences 32 . In our study, 70% (14 of 20) of anti-Ro52 positive were negative for anti-Ro60/SS-A. It remains unclear whether the definition of "anti-Ro (SS-A)" in the IPAF criteria meant a mixture of anti-Ro52 and anti-Ro60/SS-A or anti-Ro60/SS-A alone; therefore, our findings suggested that the definition of "anti-Ro (SS-A)" in the IPAF criteria should be clarified and testing anti-Ro52/TRIM21 and anti-Ro60/SS-A separately to identify the autoimmune phenotype in IIP patients.  www.nature.com/scientificreports/ www.nature.com/scientificreports/ The frequency of Raynaud's phenomenon was significantly higher in anti-Ro52-positive patients than in anti-Ro52-negative patients in our IIP cohort (P = 0.009) ( Table 3). Nearly half of the IPAF patients exhibit at least one clinical domain with Raynaud's phenomenon as the most common symptom 33,34 . In this study, all three patients with anti-Ro52 who had Raynaud's phenomenon were classified as IPAF (Supplementary Tables S1 and  S2). Thus, testing for serum anti-Ro52 might be helpful in classifying IIP patients with Raynaud's phenomenon as those meeting the IPAF criteria. Raynaud's phenomenon is associated with underlying or future development of SARD 35 but is not considered a predictor for its prognosis or development in IPAF patients 33,34 probably due to the low prevalence and short follow-up periods. Thus, the clinical significance of Raynaud's phenomenon in IPAF patients remains controversial.
Patients with anti-Ro52 have a higher frequency of rapidly progressive ILD and a higher rate of mortality than those without anti-Ro52 in SARD 13,15,16,20 . Herein, presence of anti-Ro52 was not significantly associated with overall mortality, possibly due to the heterogeneity of IIPs and the limited number of patients.
Patients with anti-ARS are associated with a unique subset characterized by clinical features, including ILDs, called anti-synthetase syndrome (AS), and several criteria of AS have been proposed 36,37 . However, AS is a "syndrome" developed for research settings, and its concept is still controversial. Recent research has reported the heterogeneity related to the prognosis and response to treatment of IIP patients with anti-ARS, wherein, certain patients were refractory to treatment with poor prognosis, while others responded well 7,38 . Patients with PM/DM positive for both anti-Ro52 and anti-ARS had severe myositis and joint impairment with a higher prevalence of ILD 14,16 . In this study, among 18 anti-ARS-positive patients, significant differences were not seen in symptoms, characteristics (Supplementary Tables S3-5), and prognosis ( Supplementary Fig. S1) related to SARD, between anti-Ro52-positive and -negative patients. However, these findings might considerably be affected by the small number of IIP patients with anti-ARS and further research is thus required.
Several limitations of this study are acknowledged. First, this study was a retrospective study with variable follow-up intervals and periods. Second, the sample size was relatively small and comprised only of Japanese individuals from two university hospitals. Third, possible missed signs and symptoms in the clinical domain of IPAF criteria may have resulted in inaccurate IPAF diagnoses because our cohort included patients enrolled before IPAF criteria was proposed. However, we routinely consulted ILD patients with rheumatologists and requested appropriate evaluations to exclude the presence of collagen vascular diseases. Fourth, although none of the patients with IIP developed any autoimmune diseases during the follow-up period, the observation period was short. It is possible that some might develop SARD in the future because ILD could precede the development of SARD in certain patients 24 .
In conclusion, the fulfillment of IPAF criteria and presence of Raynaud's phenomenon were more frequently seen in the presence than in the absence of anti-Ro52 in patients with IIP. Our findings may suggest that testing for anti-Ro52 help to identify the autoimmune phenotype and predict the development of SARD in IIP patients. Further prospective studies on a large cohort are needed to elucidate the clinical significance of anti-Ro52 in patients with IIP. Detection of serum autoantibodies. Serum samples of patients were obtained during their first visit and stored at − 20 °C until further use. For the analyses of autoantibodies, 35 S-methionine radiolabeled K562 cell extract was immunoprecipitated with IgG purified from 8 µL of human serum samples. The immunoprecipitated proteins were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously 39 . Briefly, cells were labeled with 35 S-methionine and cysteine, lysed in 0.5 M NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris (pH 7.5), 0.3% octylphenyl polyethylene glycol (IGEPAL CA-630) buffer containing 0.5 mM phenylmethylsulfonyl fluoride and 0.3 trypsin inhibitory units (TIU)/mL aprotinin, and immunoprecipitated using protein-A-Sepharose beads coated with IgG. Immunoprecipitates were then washed with 0.5 M NaCl-NET/IGEPAL CA-630 and analyzed by SDS-PAGE and autoradiography. The specificity of autoantibodies was confirmed by the use of human reference sera 39 . Antibodies to Ro52/TRIM21, histidyl-tRNA synthetase (Jo-1), and melanoma differentiation-associated protein 5 (MDA5) were tested by enzyme-linked immunosorbent assay (ELISA) as described previously 39 . All recombinant proteins were purchased from Diarect (Freiburg, Germany). Briefly, 96-well microtiter plates (Immobilizer Amino; Nunc Naperville, IL, USA) were coated with 0.5 μg/ml of recombinant protein and blocked with 0.5% bovine serum albumin (BSA)-NET/ IGEPAL CA-630 for 1 h at room temperature. Patients' sera (1:250) and alkaline phosphatase-conjugated goat anti-human IgG (1:1000; γ-chain specific; Jackson Immunoresearch, Hershey, PA, USA) diluted in 0.5% BSA-NET/ IGEPAL CA-630 were used as the sample and secondary antibodies, respectively. A standard curve was generated using serial 1:5 dilutions of a high-titer prototype serum. Optical density of samples measured at 405 nm was converted into units based on the standard curve.
Clinical data collection and analyses. Demographic data, clinical information, results of laboratory and pulmonary function tests, and analyses of bronchoalveolar lavage fluid were obtained from medical records. Physical findings were confirmed by rheumatologists when appropriate. Classification criteria of IPAF were based on the 2015 ERS/ATS Task Force research statement 5 . Although patient data (n = 281) used in this study partially overlapped with a retrospective study published earlier 6 , our research data related to anti-Ro52 are unique.

Statistical analyses.
Data are presented as the median [interquartile range] or frequency (%). Fisher's exact test was used to compare categorical variables. Comparisons between groups were made using the Mann-Whitney U test. Survival analyses were performed using the Kaplan-Meier method and the log-rank test. All analyses were conducted at a significance level of α = 0.05. All statistical analyses were performed using the STATA 16.

Data availability
The datasets used for the current study are available from the corresponding author on reasonable request.